Glioblastoma multiforme is the most frequent primary brain tumor in adults and one of the most lethal malignancies with a median survival of 12-15 months despite multi-modality treatment. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and virtually assures tumor recurrence. Thus, there is a significant unmet clinical need to develop innovative approaches to target the dispersing tumor cells for improved treatment of this disease. The non-receptor tyrosine kinase Pyk2 functions at a point of convergence to integrate signaling from cell adhesion receptors, growth factor receptors, and G protein coupled receptors that regulate cell proliferation, migration, and survival. Pyk2 is intimately involved in glioma migration and invasion in vitro and increased expression/activity strongly correlates with advancing tumor grade in patients. Silencing Pyk2 expression or inhibition of Pyk2 activity significantly extends survival in a murine glioblastoma intracranial xenograft model. Current data from both in vitro and in vivo model systems support the hypothesis that inhibition of the biological activities of Pyk2 has potential to improve the clinicl outcome of glioblastoma patients by limiting invasion and increasing sensitivity to radiation and chemotherapy. Clinical translation of tyrosine kinase inhibitors is largely directed at competitive inhibition of catalytic domains which is challenged by lack of specificity due to the significant conservation of both sequence and structure of these domains. An alternative approach to the inhibition of kinase activity is to target domains essential for the regulation of kinase activity.By mediating Pyk2 oligomerization, the amino terminal FERM domain plays a central role in the regulation of Pyk2 activity. We hypothesize that the FERM domain represents a novel target for therapeutic innovation to specifically inhibit Pyk2 activity. Currently, there are no small molecules that specifically target FERM domains. The goal of the studies proposed in this application is to conduct a high throughput screen (HTS) for the identification of small-molecule inhibitors of Pyk2 as new therapeutic agents with wide applicability. Small molecule inhibitors of Pyk2 have therapeutic application in glioblastoma, other invasive cancers, inflammation, and osteoporosis. An AlphaScreen assay will function as primary assay to identify small-molecule inhibitors of FERM domain oligomerization. This assay will be followed by cell-based assays detecting intracellular efficacy of inhibition of full-length Pyk2 activation. The hits confirmed i both assays will be prioritized using cell-based FERM dimerization and biochemical Pyk2 catalytic activity assays. The hits demonstrating efficacy in the oligomerization assay, yet inactive in kinase assay, will be validated using dry powder compounds and proceed to functional validation in glioma migration and invasion assays. A comprehensive critical path testing funnel is already in place and we anticipate rapid evaluation of hits for their suitabilityas starting points for hit-to-lead studies and for future in vivo evaluation in pre-clinical animal models.